Expression of MSTN/Smad signaling pathway genes and its association with meat quality in Tibetan sheep (Ovis aries)

Abstract Tibetan sheep is a unique breed living in Qinghai‐Tibet Plateau. Since MSTN/Smad signaling pathway plays a critical role in the regulation of muscle development, we aimed to study the mutton quality, mRNA expression of main transduction genes in the MSTN/Smad signaling pathway, and the effects of those genes on the mutton quality of Tibetan sheep in this study. Six‐month‐old Qinghai‐Tibetan sheep were selected, slaughtered, and their Longissimus lumborum, semitendinosus muscle, arm triceps, and quadriceps femoris muscle were collected. The mutton quality was evaluated, and gene expression and their association with the mutton quality were analyzed using RT‐qPCR. The results showed that the indexes of mutton quality were not significantly different between ewes and rams (p > .05) except for Warner–Bratzler shear force (WBSF) (p < .05). A total of 21 different fatty acids were detected in the muscles of Tibetan sheep, including nine types of SFA, four types of MUFA, and eight types of PUFA. The main transduction genes of the MSTN/Smad signaling pathway were found to be widely expressed in muscle tissues, but no significant differences were observed (p > .05). The correlation analysis of the main genes and mutton quality showed that MSTN was significantly correlated with redness and cooking time; Smad2, Smad3, Smad4, and TGFβRI had significant positive correlations with marbling in arm triceps; Smad3 and TGFβRII had strong negative correlations with pH24 h in Longissimus lumborum; Smad2 was negatively correlated with drip loss in Longissimus lumborum. In short, the expression level of MSTN in muscles was positively correlated with Smad2, Smad3, and Smad4 genes and negatively correlated with TGFβRII genes. Thus, the results of this study provide a theoretical basis for the regulation mechanism of the MSTN/Smad pathway on mutton quality.


| INTRODUC TI ON
Tibet has a history of more than 4000 years and Tibetan sheep are an excellent Chinese indigenous breed mainly distributed in the unique ecological environment of the Qinghai-Tibet Plateau, the world's highest and largest plateau (Lv et al., 2018). The central breeding region is located in the southwest region of the plateau.
Since Tibetan sheep live in highlands with an average altitude of >4000 m, they are well adapted to the harsh conditions, such as low oxygen, low temperatures, strong ultraviolet radiation, and severe seasonal imbalances in forage supply (Lu et al., 2021;Lv et al., 2018). This breed has unique and distinctive characteristics at the morphological, physiological, and genetic levels. Tibetan sheep graze on natural pasture all year round without supplementary feed (Jing et al., 2022). Consequently, Tibetan mutton is more popular among local consumers because it is rich in nutrition, low in cholesterol, delicious in taste, and has less odor. The Tibetan sheep industry is recently showing an extensive market prospect (Frick et al., 2017).
Genes of the MSTN/Smad signaling pathway play a crucial role in mammalian embryo development and the regeneration process of skeletal muscle (Wang et al., 2008). Smad signal transduction consists of extracellular ligands, specific receptors on the cell surface, and Smad molecules. Myostatin binds to the activin type II receptor (ActRIIB) and induces its assembly with the activin type I receptor.
Subsequently, Smad2 and Smad3, upon phosphorylation, bind to Smad4, and the whole complex is translocated to the nucleus for repressing the transcription of genes associated with muscle formation (Elkina et al., 2011). Consequently, a tight connection is established among the three reactions, which ultimately delivers extracellular signals to the nucleus for controlling the transcription of the target gene, thereby activating the associated biological effects (Thomas et al., 2000). Signaling in this pathway involves a large number of molecules like TGFβ, BMP, and hormones. Many Smad proteins like five specific glandular receptors Smad2,Smad3,Smad5, and Smad8), one universal Smad (Smad4), and two regulatory Smads (Smad6 and Smad7) are also involved in this pathway. Smad signal transduction is a dynamic process that moves between the nucleus and plasma and is mainly regulated by ligands (Altomare, 2010).
Myostatin (MSTN) can regulate quantitative characteristics of muscles, is a negative regulator of muscle growth, and is used as an indicator of meat quality and livestock yield (Bi et al., 2020). Gay et al. found that myostatin is directly involved in the control of neuromuscular interrelationships or indirectly impacts muscle size (Gay et al., 2012). Furthermore, mutations in the MSTN gene are seen in many species, such as cattle and sheep, exhibiting the "double muscle" phenomenon (Aiello et al., 2018). In 1997, researchers at Hopkins University accidentally discovered the MSTN gene in mice and confirmed it as a member of the TGFβ superfamily. Pragada et al. showed that MSTN is related to Smad3 in its function as a negative regulator of skeletal muscle growth. MSTN inhibits the expression and activation of myogenic regulatory factors through Smad3 and inhibits the differentiation of myoblasts into myotubes (Rebbapragada et al., 2003).
Earlier studies have shown that MSTN is expressed in many tissues.
Smad proteins are mediators that transmit signals from the cytoplasm to the nucleus and regulate the transcription of target genes.
Smad2 and Smad3 are the direct substrates of receptor kinases that modulate endocrine signaling (Zhu, 2006). Rehman et al. have confirmed that the expression of the Smad2 gene is higher in skeletal muscle than in smooth muscle (Hamutai et al., 2014). Lin et al. conducted a correlation analysis on gene expression in different skeletal muscle tissues of Hu sheep and found that Smad3 gene expression is affected by different tissues, growth stages, and gender (Lin et al., 2015).
Smad4 protein mediates the transcription of TGFβ responsive genes.
Smad4 protein participates in the signal transduction of all members of the TGFβ superfamily and plays a crucial role in the growth of germ cells (Ahmed et al., 2017). Bao et al. have reported that Smad4 being the central molecule of this signaling pathway is essential in testicular reproductive organs and related reproductive tissues . TGFβR I and TGFβRII are members of the transforming growth factor superfamily (TGFβ) and have been studied intensively.
Earlier studies have focused primarily on the biological functions of TGFβ family members related to medicine and embryo development.
In recent years, the research has expanded to functions such as cell growth, cloning, and genetic breeding. The TGFβ signaling pathway regulates a variety of cellular responses associated with animal growth and development, and the genes involved in this pathway have been widely studied in cattle, sheep, and pigs (Cai et al., 2019).
In summary, the MSTN gene can inhibit muscle growth and differentiation by regulating associated growth factors and proteins.
Nevertheless, the molecular composition and regulation of the MSTN/ Smad signal transduction pathway are complex due to the involvement of different regulatory factors. Consequently, the regulation mechanism and the gene expression pattern of this pathway need to be studied further. In this study, the expression profiles of major transduction genes (MSTN, Smad2, Smad3, Smad4, TGFβRI, and TGFβRII) of the MSTN/Smad signaling pathway in Tibetan sheep muscle tissues were studied by real-time fluorescence quantitative PCR, and the correlation between their expression and meat quality were evaluated. This study may provide theoretical evidence for the regulation of mutton quality by the MSTN/Smad signal pathway, which may push forward the improvement of molecular breeding methods in Tibetan sheep.

| Animals and sampling
Tibetan sheep were sampled from an abattoir (Qinghai Xiangkameiduo Animal Husbandry Co. Ltd). Six-month-old Tibetan sheep of comparable weight and health were randomly selected, followed by their slaughter (three ewes, three rams). The meat was graded, and muscle tissues, including the longissimus dorsi, semitendinosus, arm triceps, and quadriceps femoris, were collected within 20 min of slaughter, and placed in the cryopreservation tube. These tubes were quickly placed into the liquid nitrogen tank and immediately frozen at −80°C until further analysis.

| Measurement of meat quality
The fresh meat was cut into approximately 1-cm-thick slices. Meat slices were kept at room temperature, and the meat color was measured by Minolta CR 300, and color assessment was performed using CIELAB. The results were expressed as lightness (L*), redness (a*), and yellowness (b*), calibrated against a standard white tile a 5-point scale. On this scale, 1 was related to bad and 5 to a very good level of the traits. The pH of meat between the third and fourth lumbar vertebrae was determined at 45 min and 24 h after slaughter. A 5-cm section of meat was removed from the Longissimus lumborum at the first lumbar vertebra and placed on a clean rubber pad. A circular sampler with a diameter of 2-3 cm (area of approximately 5 cm 2 ) was used to cut a 1-cm-thick section of the eye muscle and immediately weighed using a weighing balance (sensitivity of 0.001 g). The sample was then placed on and covered with 18 layers of medium-speed qualitative filter paper with a high-water absorption capacity. A plastic plate was also placed on the top and bottom, followed by the application of a pressure of 35 kg for 5 min. The pressure was released, and the meat sample was weighed immediately.
Drip loss was calculated as a percentage of the amount of water loss of meat samples after being hung in a refrigerator at 4°C for 24 h to the initial weight of the samples (1 cm × 1 cm × 2 cm) (AOAC, 1984).
The Warner-Bratzler shear force (WBSF) was measured at the 12th to 13th ribs according to the method of Honikel (1998). The slices were cut perpendicular to the fiber direction with a shearing device (C-LM3B; Runhu Instrument Co., Ltd., Guangzhou, China). The odor evaluation of meat (20 min in a boiling water bath with 0.5% salt) was performed by a taste panel consisting of 20 panelists using a 5-point scale (5 = excellent, 1 = very poor). All these traits were evaluated by the panelists and data were recorded based on their preference (like/dislike) for the samples. The cooking rate of mutton was determined by weight loss after cooking the meat for 1 h in a water bath at 80°C, using the formula cooked meat rate = (weight after steaming/ weight before steaming) × 100.

| Fatty acid analysis
Fatty acids in mutton of Tibetan sheep were determined according to the method described by Juárez et al. (2008) with several modifications. Meat samples were ground in liquid nitrogen; 1.0 g of ground samples was heated with 0.7 ml of 10 mol·L −1 KOH solution and 5.3 ml of anhydrous methanol (chromatographically pure) in a water bath at 55°C for 90 min. The tubes and contents were then cooled to room temperature, mixed with 0.58 ml of a 12 mol·L −1 H 2 SO 4 solution to methylate the free fatty acids, and placed in a water bath at 55°C for 90 min. The samples were cooled to room temperature, 3 ml of hexane was added, transferred to a centrifuge tube, and centrifuged at 3000 r/min for 5 min. The supernatant was filtered with a 0.22μm nylon syringe into a short- per minute, then to 230°C at a rate of 6°C per minute, and then the temperature was maintained for 20 min. FAME peaks were identified and quantified by comparison with the retention times of a mixture of 37 FAME standards (Supelco 47,885-U; Sigma-Aldrich, St. Louis, MO, USA), which were then serially diluted to five concentrations ranging from 10 to 0.625 g L −1 . Each sample was analyzed for fatty acids in triplicate.

| Gene expression analysis
Total RNA was extracted using the TRIzol method, and concentration was determined by measuring the optical density (OD) values using a nucleic acid protein detector. RNA integrity was determined using a gel electrophoresis test. The NCBI database was used for collecting the gene sequences, and Prime5.0 was used to design and synthesize the primers:  Tables 4 and 5. After determining the annealing temperature, the Architex fluorescence quantification instrument was used to construct the amplification and dissolution curves of the corresponding genes; three independent replicates of each sample were tested.

| Statistical analysis
All quantitative data obtained in the present study were analyzed using one-way analysis of variance (ANOVA) by SPSS (version 22.0).
The mRNA expression of main transduction genes in the MSTN/ Smad signaling pathway was estimated by 2 -ΔΔCT . The multiple comparison procedure was conducted using Duncan's test, and the correlation analysis was analyzed by Pearson's two-sided test. The results were expressed as mean ± standard deviation.

| Quality analysis of Tibetan sheep meat
The shear force of ram meat was significantly higher than that of ewe meat (p < .05), but other indexes were not significantly different (p > .05). Muscle pH after 45 min of slaughtering, marbling, odor, and all other indexes were slightly higher in ram meat than in ewe meat, but no significant difference was observed (p > .05) ( Table 6). A total of 21 different types of fatty acids were detected in ewe and ram, including nine types of saturated fatty acids, four types of monounsaturated fatty acids, and eight types of polyunsaturated fatty acids. Significant differences in contents of α-linolenic acid, arachidonic acid, docosadienoic acid, and xylic acid were found between ewe and ram meat (p < .05), but other fatty acids showed no significant differences in their contents (p > .05). The proportion of oleic acid was the highest (>0.3%) in both ewe and ram meat, followed by palmitic acid (~0.2%). Lauric acid had the lowest content of 0.0006% in ram meat and 0.0004% in ewe meat (Table 7).

| Correlation analysis of gene expressions in MSTN/Smad signaling pathway and mutton quality in Tibetan sheep
The correlation analysis indicated that gene expressions and meat quality were correlated. In the Longissimus lumborum, we found a  Table 8).

| Correlation analysis of the MSTN/Smad signaling pathway-associated transduction genes in Tibetan sheep
The correlation analysis of the relative gene expression levels associated with the MSTN/Smad signaling pathway in Tibetan sheep showed that MSTN gene expression was positively correlated with Smad2, Smad3, and Smad4 genes and negatively correlated with the TGFβRII gene. Other genes also showed a positive correlation but without any significance (p > .05). The expression of TGFβRI was significantly negatively correlated with Smad3 and Smad4 gene expressions (p > .05) (Table 9).

| DISCUSS ION
Tibetan sheep in Qinghai are grazed naturally and provide meat and milk for income to the locals (Xin et al., 2011). Although there is little difference between breeds, Tibetan sheep meat is superior to Small-tailed Han sheep meat (Jiao et al., 2020). Tibetan sheep meat has good quality with pH 24 h between 5 and 6, meat cooking rate between 25% and 28%, and drip loss between 4% and 6% (Zhang et al., 2022), which are similar to the results of this study. We also observed that the shear force of ram meat was significantly higher than that of ewe meat (p < .05). Furthermore, Tibetan sheep meat contains a good proportion of diverse kinds of fatty acids (Huang, 2015). MSTN is a member of the TGFβ family, and the MSTN signaling pathway consists of TGFβRI, TGFβR II, and Smad proteins similar to the TGFβ/Smad signaling pathway that regulates skeletal muscle growth (Kollias & McDermott, 2008;Wang et al., 2016;Yao et al., 2016). Mateescu & Thonney (2005) have shown that MSTN is expressed in the semitendinosus muscle of Dorset-sired ram lamb.
The relative expression of MSTN in leg muscle is higher than that in the stomach, rumen, and cardiac muscle tissues of the Tibetan sheep (Liang et al., 2011). Xu has discovered that Smad2 and Smad4 are expressed in the heart, liver, spleen, lung, kidney, hypothalamus, pituitary, and muscle tissues of Hu sheep (Xu, 2010). Gao et al. (2016) have uncovered that the gene expressions of Smad2, Smad3, and

TA B L E 9
Relative gene expression correlation analysis of MSTN/Smad signaling pathway genes in Tibetan sheep sheep muscle tissues (Xu, 2010). TGF-βRI and TGF-ΒRII show low levels of expression in the muscle tissue of Turpan black lamb (Anwar et al., 2016). In the present study, the main MSTN/Smad signaling pathway genes of Tibetan sheep were found to be expressed in Longissimus lumborum, arm triceps, quadriceps femoris muscle, and semitendinosus muscle without any significant difference.
The correlations between the gene expressions in the MSTN/ Smad signaling pathway and meat quality of Tibetan sheep were analyzed in this study, and a significant correlation was found between gene expression in muscle tissues and meat redness, pH 24 h , drip loss, meat cooking rate, and marbling. Meat color is one of the most important physical indicators of meat quality and is directly related to the content of myoglobin (Kim et al., 2010). MSTN expression in the Longissimus lumborum of Tibetan sheep was found to be significantly correlated with meat redness. Muscle pH is an important index for determining the rate of glycolysis in muscles after slaughter (Zang, 2010). Previous research has also discovered that the decreasing pH value affects meat quality (Bertol et al., 2013). In our research, we found that Smad2 and TGFβRII genes in Longissimus lumborum had a significant correlation with pH 24 h in Tibetan sheep.
The water-holding capacity and cooking rate of meat affected both yield and product quality and were correlated with the tenderness, juiciness, and nutritional value of the meat (McKenna et al., 2005).
The expression of MSTN and Smad2 in longissimus dorsi and quadriceps femoris muscles was found to be significantly correlated with drip loss and the cooking rate of meat. Marbling and tenderness determine the juiciness and freshness of meat, which are important indicators of meat quality and taste (Liu, 2007). This study concluded that Smad2, Smad3, Smad4, and TGFβRI genes in arm triceps and semitendinosus muscle were significantly correlated with marbling. The MSTN/Smad signaling pathway plays an important role in the regulation of postnatal skeletal muscle growth, development, and repair in mammals, such as sheep and cattle (Kollias & McDermott, 2008), and MSTN inhibits skeletal muscle growth and development (Elliott et al., 2012). When TGFβ binds to TGFβRII, the  , which was inconsistent with the findings of this study. Hence, specific reasons and mechanisms leading to these correlation differences require further experimentation.

| CON CLUS IONS
In conclusion, this study demonstrated an association between

ACK N OWLED G M ENTS
We would like to thank Qingwei Wu for his experimental help. We are grateful to Yuting Deng and Shengwei Jin for their assistance in sample collections. We thank Edited and one of the native Englishspeaking editors for their help in English proofreading and editing the final manuscript.

CO N FLI C T O F I NTE R E S T
We certify that no financial organization has a conflict of interest concerning the material discussed in this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S A PPROVA L
All animal procedures were carried out under the guidelines of the